The present invention, in some embodiments thereof, relates to pharmaceutical agents and, more particularly, but not exclusively, to digoxin and digitoxin derivatives exhibiting selective inhibition of α2-containing isoforms of Na,K-ATPase, and uses thereof to reduce intraocular pressure (IOP), and/or as cardiotonic agents in a subject in need thereof.
Glaucoma is a disease leading to irreversible blindness. Control of intraocular pressure (IOP) is the mainstay of current therapy of glaucoma, and is achieved by various drugs, such as β-blockers, prostaglandin analogues, α2 adrenergic receptor agonists, cholinergic agonists and carbonic anhydrase inhibitors given topically or systemically. The topical route is preferable, provided the drug effectively permeates the cornea, because this minimizes systemic side-effects. Despite the selection of drugs available, uncontrolled IOP in many patients eventually makes surgical intervention necessary. Thus, fresh approaches to drug treatment of glaucoma are highly desirable.
The Na,K-ATPase is the motor for production of the aqueous humour (bodily fluid) in the ciliary body epithelium and, in principle, inhibition of the Na,K-ATPase can suppress the production of aqueous humour, and control IOP. Control of: IOP is the mainstay of glaucoma therapy; however, the available drugs suffer from a variety of shortcomings, particularly due to systemic adverse effects and low therapeutic index. Previously, intra-venous digoxin, a classical inhibitor of the Na,K-pump, typically used primarily to treat congestive heart failure, was considered for this role but was discarded due to systemic toxicity.
Isoforins of the Na,K-ATPase ion pump consists of α and β subunits (α/β) and accessory FXYD regulatory subunits. There are four isoforms of the α1 subunit (α1-4) and three isoforms of the β subunit (β1-3) expressed in a tissue-specific fashion. The α1 isoform is the common isoform that maintains Na and gradients in all tissues, α2 is expressed mainly in muscle and astrocytes, and α3 is expressed mainly in nerve cells. For example, human heart expresses al (about 70%) and both α2 and α3 isoforms (about 30%) and β1.
The ciliary epithelium in the eye is a functional syncytium consisting of apical pigmented cells (PE) oriented towards the blood and baso-lateral non-pigmented (NPE) cells oriented towards the anterior chamber of the eye. It is known that the primary Na,K-ATPase isoform of the PE is α1β1 while that of the NPE is αβ3. The Na,K-ATPase in the NPE powers the production of the aqueous humor and controls intraocular pressure.
Thus, in principle, topically applied α2-selective cardiac glycosides that penetrate the intact eye and reach the ciliary epithelium could effectively reduce IOP. A potential advantage of topical application could be that systemic toxic effects typical of cardiac glycosides should be minimal.
Another possible application of an α2-selective cardiac glycoside could be as an effective cardiotonic drug, with reduced cardiotoxicity, compared to known drugs such as digoxin. Digitalis drugs such as digoxin have been used to treat heart failure for over two hundred years but are dangerous drugs with multiple side effects. There is now good evidence that selective inhibition of α2 is especially effective in enhancing cardiac excitation-contraction coupling and mediating cardiac glycoside-mediated positive inotropy. Inhibition of α2, which is a minor isoform and is located largely in T-tubules, may mediate the positive cardiotonic effects, but α2-selective cardiac glycosides should only minimally inhibit α1, located primarily in the outer sarcolemma membrane, and thus avoid cellular Ca overload, the hallmark of cardiac toxicity.
The isoform selectivity of a large number of known cardiac glycosides has been previously studied, using the yeast P. pastoris expressing Na,K-ATPase isofoinis (α1β1, α2β1, α3β1), and purified detergent-soluble isoform complexes of Na,K-ATPase [Cohen E. et al., 2005, J Biol Chem, 280(17), pp. 16610-16618; Flaviv H, et al., 2007, Biochemistry, 46(44), pp. 12855-12867; Lifshitz Y, et al, 2007, Biochemistry, 46(51), pp. 14937-14950; Mishra N K, et al., 2011, J Biol Chem, 286(11), pp. 9699-9712; and Kapri-Pardes E, et al., 2011, J Biol Chem, 286(50), pp. 42888-42899].
Dissociation constants, KD, for digitalis glycosides, digoxin and digitoxin, measured by 3H-ouabain displacement assays in membranes, showed moderate selectivity (3-4-fold) for α2/α3 over α1. By contrast to the digitalis glycosides, the KD of ouabain showed some preference for α1 over α2 and similar Ki values for all three isoforms. In assays of inhibition of Na,K-ATPase activity, measured with the purified isoform protein complexes, digoxin and digitoxin showed 3-4-fold lower Ki (inhibition) values for α2 compared to α1, with α3 more similar to α1. No aglycones of any cardiac glycosides tested showed isoform selectivity. For digoxin derivatives, with one to four digitoxose moieties the maximal α2/α1 selectivity was found for digoxin itself, with three digitoxose sugars [Katz, A. et al., J Biol Chem, 2010, 285(25), pp. 19582-19592].
Based on recent studies [Laursen, M. et al., Prot Nati Acad Sci USA, 2015, 112(6):1755-60], it was inferred that the sugar moiety of digoxin likely determines isoform selectivity, which is generally consistent with recent structures of Na,K-ATPase with bound ouabain, bufalin or digoxin. The unsaturated lactone ring and steroid portion of ouabain are bound between trans-membrane segments M1, M4, M5 of the α subunit, in which there are no amino-acid differences between isoforms. Assuming that the aglycones of all cardiac glycosides bind similarly, the implication is that isoforms cannot discriminate between any of the aglycones, as found experimentally. By contrast, the sugar is bound near extracellular loops, where there are a number of amino-acid differences between the isoforms. These residues might interact with the sugars of bound digoxin in an isoform-selective way.
Additional background art include WO 2015/029035, WO 2007/079128, WO 2010/053771, U.S. Patent Application No. 2005/0032138 and U.S. Pat. Nos. 7,888,059 and 7,851,145; these documents are hereby incorporated by reference.